More than 10% of the world population suffer from allergy to grass pollen. Here we describe the development of a vaccine based on recombinant hypoallergenic hybrid molecules which were constructed out of elements derived from the four major timothy grass pollen allergens Phl p 1, Phl p 2, Phl p 5, and Phl p 6 for the treatment of grass pollen allergy. Codon-optimized synthetic genes encoding building blocks and combinations of the four allergens were designed according to epitope mapping studies and structural data and subsequently expressed in Escherichia coli. Seventeen recombinant hybrid molecules were purified by affinity chromatography and evaluated regarding expression, purity and fold, solubility, and reduced allergenic activity. Four hypoallergenic hybrid molecules consisting of reassembled elements of the four grass pollen allergens were identified which upon immunization in different animal models induced IgG antibodies blocking IgE recognition of the grass pollen allergens by allergic patients. These hypoallergenic hybrid molecules represent safe vaccines for immunotherapy of grass pollen allergy.
IgE-mediated allergies represent a worldwide health problem with increasing prevalence (1). The hallmark of allergic disease is the production of IgE antibodies specific for environmental allergens, mainly from pollen, mites, animal dander, and moulds (1). Allergic symptoms occur, when receptor-bound IgE on mast cells or basophils is cross-linked by multivalent allergen, leading to the release of inflammatory mediators (2). In addition, IgE-facilitated presentation via FcεRI and FcεRII on antigen presenting cells strongly enhances the activation of allergen-specific T cells contributing to T cell mediated allergic inflammation (3, 4). Allergen-specific immunotherapy currently represents the only causative treatment of allergies with a long-term effect, although its success is impaired by the use of crude allergen extracts (5, 6). These preparations contain allergenic and non-allergenic material in varying amounts, whereby the presence of biologically active compounds increases the risk of anaphylactic side effects. In addition, the lack or poor immunogenicity of clinically relevant allergens reduces the efficacy of extract-based vaccines (7). Substantial progress has been made in the field of allergen characterization during the last 20 years through the application of immunochemical and molecular biological techniques. Today, the most common and important allergens have been characterized regarding their structure and immunological properties. Recombinant allergens closely resembling the properties of the natural allergens have been produced and can now be used for the diagnosis and therapy of allergy (5). In addition it has been shown that allergen derivatives with beneficial immunological properties can be engineered (8). Modified variants of allergens with reduced allergenic activity have been generated, in order to avoid IgE-mediated side effects in the course of immunotherapy and recombinant Bet v 1-derived fragments with strongly reduced IgE-binding capacity have already been used in a clinical trial (9). Hybrid molecules consisting of combinations of different allergens have been shown to increase the immunogenicity of their single components (10-12).
Linhart et al. (Ref. 21) prepared a hypoallergenic hybrid molecule with increased immunogenicity by combining hypoallergenic derivatives of the two major grass pollen allergens Phl p 2 and Phl p 6, namely a Phl p 2 mosaic molecule and a deletion mutant of Phl p 6, which was not surprising in light of previous data (Ref. 15, 17).
The present inventors now have found that a combination of the hybrid technology and the mosaic technology does not in all cases lead to hypoallergenic molecules. Surprisingly, it has been observed that two fusion polypeptides which consisted of the same fragments but in a different order exhibited very different IgE reactivities. The present invention therefore provides a method for identifying polypeptides that have hypoallergenic properties and can serve as a potential vaccine.